Effect of photoperiod and allatectomy on reproduction and (second) diapause induction in post-diapause adult females of the Colorado potato beetle,Leptinotarsa decemlineata Say

Summary

The effect of photoperiod and allatectomy on the rates of oviposition and juvenile hormone (JH) synthesis was studied in female beetles after the break of adult diapause. The results indicate that after diapause, photoperiod has no effect on oviposition rates. However, the photoperiodic effect on diapause induction is restored within a few days after the break of the first diapause. Allatectomy performed within one day after diapause did not induce a second diapause, whereas about 40% of the beetles allatectomized after 3 or more days responded by entering a second diapause. Oogenesis in the operated animals was never completely prevented under long and short day conditions. These preliminary results suggest a change in the sensitivity of post-diapause beetles to photoperiodic and endocrine manipulation as compared with pre-diapause insects.     

MOULTING HORMONE ACTIVITY IN THE ADULT COLORADO POTATO BEETLE,LEPTINOTARSA DECEMLINEATA SAY IN RELATION TO REPRODUCTION AND DIAPAUSE

Ecdysteroid levels in Leptinotarsa decemlineata were measured by radioimmunoassay (RIA), using an antiserum with a 10 times higher affinity for ecdysterone than for ecdysone and polyethylene glycol as a means for separating free from bound hormone. In reproducing males, raised under long day conditions (LD: 16 h photophase, 25°C) there was a peak of 54 ng/g at day 3–4, declining thereafter to very low values. In LD-females, 50 ng/g was found at day 2 (just before the onset of vitellogenesis), decreasing to 20 ng/g at day 5 (start of oviposition) and increasing again thereafter to 95 ng/g at day 14. In prediapausing animals, raised under short day conditions (SD: 10 h photophase, 25°C) the level was practically the same in both sexes and it was significantly higher than in reproducing animals. In both sexes a peak of about 96 ng/g was found from day 2 to 4, thereafter the level dropped quickly. During diapause the level increased slowly and in 9 month diapausing adults it was about 75 ng/g. This is the first time that high ecdysteroid levels are reported in relation to diapause induction in an adult insect, and the first time that high levels are found in adult males, indicating that the ovary cannot be the only site of synthesis of moulting hormone. The content during metamorphosis was also determined, mainly as a test for our RIA method. Hitherto there was no indication whatsoever that moulting hormone (MH) might be present and play a role in the adult stage in this insect. Our results make necessary a reappraisal of the hormonal regulation of oogenesis and induction of diapause in the Colorado beetle. The nature of the ecdysteroids present in the adult stage, their site of synthesis, their metabolism and physiological significance remain to be determined.     

EVOLUTIONARY ENDOCRINOLOGY OF JUVENILE HORMONE ESTERASE: FUNCTIONAL RELATIONSHIP WITH WING POLYMORPHISM IN THE CRICKET,GRYLLUS FIRMUS

The existence, nature, and physiological consequences of genetic variation for juvenile hormone esterase (JHE) activity was studied in the wing-polymorphic cricket, Gryllus firmus. Hemolymph (blood) JHE activity was sixfold lower in nascent short-winged (SW) females, relative to nascent long-winged (LW) females during the last juvenile stadium (stage). Morph-associated genetic variation for JHE activity had two causes, variation in loci: (1) regulating whole-organism enzyme activity; and (2) controlling the degree to which JHE is secreted into the blood Reduced JHE activity in nascent SW-selected individuals was associated with reduced in vivo juvenile hormone catabolism. This suggests that variation in JHE activity during juvenile development may have important physiological consequences with respect to the regulation of blood levels of juvenile hormone and consequent specification of wing morph. This is the first definitive demonstration of genetic variation for hormonal metabolism in any insect and a genetic association between hormone metabolism and the subsequent expression of morphological variation (wing morph). However, we have not yet firmly established whether these associations represent causal relationships In contrast to the clear association between JHE activity and wing morph development, we observed no evidence indicating that variation in JHE activity plays any direct or indirect role in causing the dramatic differences in ovarian growth between adult wing morphs. Variation in JHE activity also does not appear to be important in coordinating the development of wing morph with the subsequent expression of reproductive differences between adult morphs. Finally genetic variation for the developmental profiles of JHE activity during juvenile and adult stages are remarkably similar in three Gryllus species. This suggests that genetic correlations between JHE activities during different periods of development, which underlie these activity profiles, have been conserved since the divergence of the three Gryllus species.     

不同寄主植物上马铃薯甲虫种群生长发育的比较研究

马铃薯甲虫Leptinotarsa decemlineata (Say)是马铃薯Solanum tuberosum等茄科作物上的一种毁灭性害虫。为了探明寄主植物对新疆马铃薯甲虫种群生长发育的影响, 本研究通过非选择性试验测定了马铃薯、 茄子Solanum melongena和番茄Lycopersicon esculentum 3种栽培寄主及野生寄主中亚天仙子Hyoscyamus pusillus对马铃薯甲虫种群生长发育、 存活、 繁殖及生命表参数的影响。结果表明： 马铃薯甲虫幼虫虽然能够取食番茄, 但幼虫的发育历期长、 存活率低且蛹不能羽化, 表明番茄不是新疆马铃薯甲虫种群的适宜寄主。马铃薯、 茄子、 中亚天仙子3种寄主植物对该虫卵、 幼虫、 蛹的存活率和发育历期及成虫产卵前期没有显著影响, 但对蛹重和繁殖力影响显著。取食不同寄主植物后, 该虫蛹重和繁殖力从大到小的次序为： 马铃薯＞茄子＞中亚天仙子; 种群的净增殖率(R₀)、 内禀增长率(r_m)和种群趋势指数(I)从大到小依次为： 马铃薯＞茄子＞中亚天仙子。因此马铃薯为新疆马铃薯甲虫种群的最适宜寄主, 其次为茄子和中亚天仙子, 而番茄为不适宜寄主。     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

保幼激素类似物对家蚕丝腺与脂肪体蛋白质合成的调节作用

应用示踪原子法,研究了家蚕Bombyx mori 5龄幼虫丝腺与脂肪体细胞内蛋白质合成的变化规律及保幼激素类似物(JHA738)的调节作用。从5龄初到龄末,家蚕丝腺细胞内蛋白质合成持续升高,5龄中期、后期的蛋白质合成活性分别是前期的1.60倍和2.86倍;全龄出现2个合成高峰,一个是在5龄72h,为细胞固有蛋白质合成高峰,另一个是在5龄192h,为丝蛋白合成高峰。脂肪体细胞内蛋白质合成作用呈现脉冲式的变化。在5龄前期和中期用JHA处理家蚕(剂量为4μg/条),对丝腺细胞固有蛋白质合成和脂肪体细胞蛋白质合成均表现出抑制作用,而对丝蛋白合成则表现出促进作用。本实验结果为进一步阐明JHA增丝机理提供了直接证据。     

褐飞虱的交配行为及与保幼激素滴度的关系

受5龄末期和羽化初期较高保幼激素滴度的影响,褐飞虱Nilaparvata lugens(Stal)短翅型成虫性成熟比长翅型成虫早40～50h。5日龄前短翅型雌成虫的交配成功率较高,雄成虫的交配竞争能力较强;1、2日龄的长翅型雌成虫不交配,5日龄后长翅型成虫的交配能力渐渐超过短翅型成虫。实验种群的交配持续时间为(70.38±41.77)s;交配成功率为51.63%;雌、雄虫均可重复交配。一日中0～4h和10～14h交配成功率较高。     

促黄体素释放激素类似物和多巴胺对鲤鱼幼鱼和性成熟雌鱼生长激素分泌的作用

以性腺未发育的５～６月龄幼鲤和１龄性成熟雌鲤为材料,研究年龄对促黄体素释放激素类似物和多巴胺能药物刺激的鲤鱼ＧＨ分泌的影响。结果表明：５～６月龄幼鱼血清基础ＧＨ水平明显低于１龄性成熟鱼;５～６月龄幼鱼对ＬＨＲＨＡ、ＤＡ和ＡＰＯ刺激的ＧＨ分泌的反应性也明显低于１龄性成熟鱼;ＤＡ和ＡＰＯ在５～６月龄幼鱼和１龄性成熟鱼都不能增强ＬＨＲＨＡ对ＧＨ分泌的刺激作用;ＤＯＭ不影响５～６月龄幼鱼和１龄性成熟鱼的血清ＧＨ水平。     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

CHARACTERIZATION AND MOLECULAR PHYLOGENYOF A PROTEIN KINASE cDNA FROM THE DINOFLAGELLATE GONYAULAX (DINOPHYCEAE)1

Degenerate primers corresponding to conserved protein kinase motifs were used to amplify potential kinase DNA fragments from a Gonyaulax polyedra Stein cDNA library using PCR. One PCR fragment, potentially encoding a CAMP-dependent protein kinase, was used as a probe to isolate a near full-length cDNA from the library. The nucleic acid sequence of the entire cDNA clone had a high homology to the catalytic subunit of cAMP-dependent protein kinase (cAPK subfamily and affiliated members. Northern blot analysis showed that the corresponding mRNA had a size (about 1.4 kb) and a relative high abundance consistent with a cAPK homologue. Southern blot analysis showed that while there are roughly 30 copies of the kinase gene per genome, the pattern of restriction fragments is inconsistent with the hypothesis of a large gene family. Phylogenetic analyses comparing the deduced amino acid sequence from the Gonyaulax cDNA with other cAPK sequences place Gonyaulax close to the slime mold Dictyostelium discoideum. This is the first phylogenetic analysis of dinoflagellates based on protein sequence, and the results are in agreement with similar analyses based on rRNA sequences.     

CONDITIONAL PRODUCTION OF A FUNCTIONAL FISH GROWTH HORMONE IN THE TRANSGENIC LINE OF NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Plasmid phr‐YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a RUBISCO SSU 2 promoter, was transferred into the protoplast of marine microalga Nannochloropsis oculata (Droop) D. J. Hibberd by electroporation. Four transgenic clones were obtained in which the transferred phr‐YPGHc was integrated into the genome and existed stably at least until the 50th generation. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in the amount of 0.42 to 0.27 μg · mL^?1 from the 50 mL of medium. We incubated artemia with the wildtype and transgenic N. oculata for 6 h and then fed these microalgae‐treated artemia to red‐tilapia larvae. After feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater (i.e., statistically significant: P < 0.05) than that of larvae that were fed artemia incubated with nontransgenic microalgae: 316% versus 104% in weight gain, and 217% versus 146% in body length increase, respectively. Therefore, the N. oculata enables production of functional GH, and we propose that it might be an excellent bioreactor material.     

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF PROTEASES IN LARVAL PREPARATIONS OF THE CEREAL LEAF BEETLE (Oulema melanopus,CHRYSOMELIDAE, COLEOPTERA)

We determined some biochemical properties of Oulema melanopus larval gut proteases. We found adult midgut enzyme preparations yielded results similar to whole‐larval preparations, permitting studies of the very small whole‐larval preparations. Protein preparations were analyzed using FITC–casein as a substrate. Acidic pH is optimal for proteolytic activity (range 3.0–4.0). Cysteine protease activity increased at acidic pH and in the presence of β‐mercaptoethanol. Protease activities at all pH values were maximal at 45°C. Enzyme activity in larval preparations was inhibited by addition of Fe²⁺, Ca²⁺, Mg²⁺, Zn²⁺, and K⁺ (10 mM). Fe²⁺ and Zn²⁺ significantly decreased enzyme activity at all pH values, Ca²⁺ and Mg²⁺ at pH 6.2 and Mg²⁺ at pH 4.0. Inhibitors, including pepstatin A, showed the greatest inhibition at pH 4.0; phenylmethylsulfonyl fluoride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone at pH 6.2; and phenylmethylsulfonyl fluoride, N_α‐tosyl‐l‐lysine chloromethyl ketone hydrochloride, N‐p‐tosyl‐l‐phenylalanine chloromethyl ketone, trans‐epoxysuccinyl‐l‐leucylamido‐(4‐guanidino) butane at pH of 7.6. Inhibition assays indicated that cysteine, aspartyl (cathepsin D), serine (trypsin, chymotrypsin‐like) proteases and metalloproteases act in cereal leaf beetle digestion.     

丘脑下部促黄体素释放激素类似物(LHRH-A)对文昌鱼精子发生的影响:组织学及其超微结构的研究

本文报道将LHRH-A注射到文昌鱼体腔,可促使其精巢发育的实验结果。发育的精巢占实验动物总数的87％,没有变化者占17％。组织学和电镜观察指出LHRH-A可激发A型精原细胞分裂为B型精原细胞,促使初级精母细胞发育为次级精母细胞和精细咆。另外,LIRH-A有促使精于的释放的作用。 我们认为LHRH-A可激发文昌鱼的生精活动。     

草鱼生长激素非竞争式酶联免疫吸附测定法的建立及鉴定

应用草鱼生长激素（ｇｃＧＨ）单克隆抗体及多价兔抗血清建立了草鱼ＧＨ非竞争式酶’联免疫吸附测定ＥＬＩＳＡ系统。用正辛酸法对腹水单抗进行了分离纯化,获得了高纯度的单抗制备物。聚丙烯酸胺凝胶电泳表明纯化的单抗由分子量分别为５５ｋＤ和２５ｋＤ的两条蛋白带组成。用纯化单抗铺底,用兔抗血清作后续抗体建立了一种测定草鱼ＧＨ的非竞争式双抗夹心ＥＬＩＳＡ方法。交叉试验表明该测定系统只与草鱼ＧＨ和基因重组鲤生长激素（ｒｃＧＨ）具有剂量依存的结合反应,而与大马哈鱼生长激素（ｓＧＨ）、牛生长激素（ｂＧＨ）、大马哈鱼促性腺激素（ｓＧｔＨ）、及黑鲢促性腺激素（ｂｓｃＧｔＨ）等均无交叉反应。该 ＥＬＩＳＡ方法的灵敏度可达０．８ｎｇ／ｍｌ,组内变异系数为 ５．９ ％,组间变异系数为７．６％,回收率达９０％以上。初步应用表明,鲤和团头鲂垂体抽提液、草鱼血清、鲤血清及鲫血清在该测定系统中有剂量依存的反应曲线,而大口鲶、黄颡鱼、中华鲟及黄鳝鱼垂体抽提液及大口鲶、胡子鲶和罗非鱼血清在该测定系统中没有交叉反应。     

ULTRASTRUCTURE OF THE BASAL APPARATUS AND PUTATIVE VESTIGIAL FEEDING APPARATUSES IN A QUADRIFLAGELLATE EUGLENOID (EUGLENOPHYTA)1

The flagellar apparatus and presumptive vestigial feeding apparatuses of a cold-water, photosynthetic, quadriflagellate euglenoid is described. The organism possesses two similar sets of flagella each consisting of one short and one long flagellum. Each pair of flagella is associated with three microtubular roots for a total of six roots in the basal apparatus. At the level of the ventral basal bodies, each intermediate root is nine-membered, while the ventral roots are composed of eight to nine microtubules. Only one of the ventral roots lines the single microtubule reinforced pocket. A four-membered dorsal root attaches to each dorsal basal body, and at the level of the reservoir each gives rise to a dorsal band. An additional bundle of microtubules, not arising from the microtubular roots of the basal apparatus, begins posterior to the basal apparatus as a small group of a few microtubules and extends anteriorly on the right ventral side of the reservoir ending at the canal. At the level of the stigma, the microtubules are organized into a multi-layered bundle that continues to increase in size and eventually splits to form two bundles at the level of the canal. We postulate that these bundles may represent the remnants of a rod-and-vane-type feeding apparatus like that found in many phagotrophic euglenoids.     

楝星天牛胸部摩擦和鞘翅振动发声及其声学特性的研究*

楝星天牛Anoplophora horsfieldi(Hope)成虫可借两种不同的方式发声： 1.胸部摩擦发声;2.鞘翊振动发声。胸部摩擦发声可连续进行,具有抗拒敌害和种内通讯的生物学意义;鞘翅振动发声为断续产生,受外界刺激而诱发,主要与抗拒敌害有关。测定表明,胸部摩擦声由多个音组连续组成,每一音组的持续时间为1100-1 6000ms,依次由抬头声音节(持续384±22ms,含有250-350个脉冲列)、间隔1(270-600ms)、低头声音节(382±16ms,250-350个脉冲列)和间隔2(80-360ms)组成。 抬头声音节的功率谱由基本音及其分音组成,基本音为信号能量的主要部分,其主峰频率为742±30Hz(雄)和603±34HZ(雌)。鞘翅振动声由间隔不等的,7-9个脉冲列组成,脉冲列的振幅较大、频率较高,持续约7-10ms。功率谱图上虽然脉冲列的频率分布于0-3100Hz,但能量主要集中在850-1050Hz内。与胸部摩擦声相比,鞘翅振动声的能量更高、释放更突然,是一种更为有效的拒敌性行为。     

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

In our previous study, we generated a strain of 19‐P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780‐bp stem. We utilized this RNAi‐induced strain to uncover RNAi‐related genes. Random insertional mutagenesis was performed to generate tag‐mutants that show a RNAi deficient phenotype. The 92‐12C is one such tag‐mutant, which bears a 14‐kb deletion in chromosome 1. Complementation of 92‐12C revealed that a protein gene, including a Cys‐Cys‐Cys‐His‐type zinc finger motif and an ankyrin repeat motif, is essential for effective RNAi in Chlamydomonas reinhardtii (Dangeard). BLAST analysis revealed that the zinc finger protein is homologous to an mRNA splicing‐related protein of other species. Therefore, one of the probable scenarios is that mRNA coding for RNAi‐related proteins cannot be properly spliced, which causes RNAi deficiency in the 92‐12C tag‐mutant.